Human kappa opioid receptor, nucleic acids and uses thereof

ABSTRACT

Novel peptides having human kappa opioid receptor activity, genetic material for expressing same, any recombinant cell expressing said polypeptides, and use thereof.

The present invention relates to new polypeptides and the geneticmaterial permitting their expression. More particularly, it relates tonew polypeptides having a human kappa opioid receptor activity.

Opioid receptors are membranous receptors of the nervous system whichmodulate the analgesic and psychotropic properties of alkaloid drugs ofthe morphine type (Brownstein, 1993, Proc. Natl. Acad. Sci. USA, Vol.90, 5391). Pharmacological studies have demonstrated the existence ofthree subtypes of receptors, called mu, delta and kappa, which differ bytheir capacity to bind to different opioid ligands (Goldstein et al.,1989, Mol. Pharmacol., Vol. 36, 265-272) and by their distribution inthe organism (Mansour et al., 1987, J. Neurosci., Vol. 7, 2445).Recently, three opioid receptors have been cloned in rodents. Thepharmacological profile of these three cloned receptors, expressedtransitorily in Cos cells, indicate that there is a delta receptor, a mureceptor and a kappa receptor. Analysis of their primary structureconfirms that they are part of the family of receptors coupled to the Gproteins, which have a putative topology with seven transmembranaldomains (Bockaert, 1991, Curr. Op. Neurobiol., Vol. 1, 32).

The present invention comprises the elucidation, isolation and molecularcharacterization of the human kappa opioid receptor. It comprisesparticularly the isolation and sequencing of the gene coding for thisreceptor, in the construction of recombinant strains permitting theexpression of functional receptors, and in the elaboration of testspermitting the isolation of compounds active on these receptors andhaving desirable therapeutic properties. The DNA sequences of theinvention also permit the provision of probes capable of detecting inbiological specimens any irregularity in the expression of a kappaopioid receptor (non-expression, mutation, polymorphism, etc.). Theseprobes are also usable for cloning by hybridization of any other cDNAcoding for an opioid receptor, from tissues of diverse origins andparticularly of human origin.

A first object of the invention therefore resides in a nucleotidesequence coding for a polypeptide having a human kappa opioid receptoractivity.

More preferably, the nucleotide sequence according to the invention isselected from:

(a) all or a portion of the nucleotide SEQ ID No. 1 or of itscomplementary strand,

(b) any sequence hybridizing with a sequence (a) and coding for apolypeptide having human kappa opioid receptor activity, and,

(c) sequences derived from sequences (a) and (b) by degeneration of thegenetic code.

The different nucleotide sequences of the invention can be of artificialorigin or not. They can be genome sequences, cDNA sequences, RNAsequences, hybrid sequences or synthetic or semi-synthetic sequences.These sequences can be obtained for example by screening DNA banks (cDNAbank, DNA genome bank) by means of probes developed on the basis of thesequence SEQ ID No. 1. Such banks can be prepared from cells ofdifferent origins by conventional bimolecular techniques known to thosein the art. The nucleotide sequences of the invention can also beprepared by chemical synthesis, particularly according to thephosphoramidites method, or again by mixed methods including chemical orenzymatic modification of sequences obtained by scanning banks.

The nucleotide sequences of the invention can be used for the productionof kappa opioid polypeptides. The term kappa opioid polypeptidedesignates any polypeptide having a kappa opioid receptor activity, andany fragment or derivative of such a polypeptide. For the production ofkappa opioid polypeptides, the coding portion for said polypeptide isgenerally placed below the signal control permitting its expression in acellular host. The choice of these signals (promoters, terminators,etc.) can vary as a function of the cellular host utilized. To this end,the nucleotide sequences of the invention can be a part of a vector,which can have autonomous or integrated replication. More particularly,the autonomous replication vectors can be prepared by using autonomousreplication sequences in the selected host. Acting on the integrativevectors, these latter can be prepared for example by using sequenceshomologous to certain regions of the genome of the host, permitting byhomologous recombination, the integration of the vector. The cellularhosts usable for the production of the opioid polypeptides of theinvention by recombinant means, can be either the eucaryote hosts orprocaryote hosts. Among the eucaryote hosts which are suitable, can becited animal cells, yeasts, or mushrooms. In particular, for yeasts, canbe cited the yeasts of the type Saccharomyces, Kluyveromyces, Pichia,Schwanniomyces, or Hansenula. As to the animal cells, can be cited thecells COS, CHO, C127, NIH-3T3, etc. As to mushrooms, can be cited moreparticularly Aspergillus ssp. or Trichoderma ssp. As procaryote hosts,it is preferred to use the following bacteria: E.coli, Bacillus, orStreptomyces.

The nucleotide sequences of the present invention are also usable in thepharmaceutical field, either for the production of anti-directionalsequences usable in the field of gene therapy, or for the production ofprobes permitting the detection, by hybridation experiments, of theexpression of opioid receptors in biological specimens and theelaboration of genetic anomalies (polymorphism, mutations) or ofaberrant expressions.

The inhibition of the expression of certain genes by anti-directionalsequences is thought to be a promising strategy in the control of theactivity of a gene. The anti-directional sequences are sequences whosetranscription product is complementary to the strand coding a givengene, and is thus capable of specifically hybridizing with thetranscribed mRNA, inhibiting its translation into protein (EP140 308).The invention thus has for its object anti-directional sequences capableof inhibiting at least partially the production of kappa opioidpolypeptides such as defined above. Such sequences can be constituted byall or a portion of the nucleotide sequences defined above. It relatesgenerally to sequences or to fragments of sequences complementary to thesequences coding for peptides of the invention. Such sequences can beobtained from the sequence SEQ ID No. 1, by fragmentation, etc. or bychemical synthesis.

As indicated above, the invention also permits the provision ofnucleotide probes, synthetic or not, capable of hybridizing with thenucleotide sequences defined above which code for the opioidpolypeptides of the invention, or with the corresponding mRNA. Suchprobes can be used in vitro as diagnostic tools, for the detection ofthe expression of a kappa opioid receptor, or for the elaboration ofgenetic anomalies (poor splicing, polymorphism, point mutations, etc.).Given the multiple activities of the endogenous ligands of the opioidreceptors, the probes of the invention can thus permit identifyingneurological, cardiovascular or psychiatric afflictions. These probescan also be used for the elaboration and isolation of homologous nucleicacid sequences coding for opioid polypeptides as defined above, fromother cells sources and preferably from cells of human origin. Theprobes of the invention generally comprise at least 10 bases, and theycan comprise up to all of the sequence SEQ ID No. 1 or its complementarystrand. Preferably, the probes are, before their use, marked. To dothat, different techniques known to those in the art can be used(radioactive marking, enzymatic marking, etc.). The conditions ofhybridation under which these probes can be used are indicated in thegeneral cloning technique hereinafter as well as in the examples.

The invention also has for its object any polypeptide resulting from theexpression of a nucleotide sequence as defined above. Preferably, it isa polypeptide comprising all or a portion of the polypeptide sequenceSEQ ID No. 1 or of a derivative of the latter.

In the meaning of the present invention, the term derived designates anymolecule obtained by modification of the genetic and/or chemical natureof the polypeptide sequence SEQ ID No. 1 and retaining a kappa opioidreceptor activity. By modification of genetic and/or chemical activityis meant any mutation, substitution, deletion, addition and/ormodification of one or several residues. Such derivatives can begenerated for different purposes, such as particularly to augment theaffinity of the peptide for its ligand or ligands, to improve itsproduction level, to increase its resistance to proteases, to increaseand/or modify its activity, or to give it new pharmacokinetic and/orbiological properties. Among the derivatives resulting from an addition,can be cited for example the chimeral polypeptides comprising asupplemental heterological portion connected at one end.

Preferably, the polypeptides of the invention are polypeptides havingthe capacity to bind dynorphine and the derivatives of the type ofprodynorphine. More preferably, they have the capability of bindingagonists of dynorphine such as U-50,488, ethylcetocyclasoncine andbremozacine, and the antagonists of the dynorphine such asnorbinaltorphimine. According to a preferred embodiment, thepolypeptides of the invention are adapted to be recognized by antibodiesrecognizing the complete peptide sequence of SEQ ID No: 2. Suchantibodies can be generated by any technique known to those in the art,by using as antigens the polypeptides described in the presentapplication.

As indicated in the examples, these polypeptides can be expressed indifferent cell types to form functional opioid receptors.

The polypeptides of the invention can be obtained by expression in acellular host of a nucleotide sequence such as described above, bychemical synthesis, on the basis of the SEQ ID No. 1 sequence, by usingtechniques known to those in the art, or by a combination of thesetechniques.

Another object of the invention concerns recombinant cells capable ofexpressing at their surface a polypeptide having a human kappa opioidreceptor activity. These cells can be obtained by introduction of anucleotide sequence as defined above, then by culturing said cells underconditions for the expression of said sequence.

The recombinant cells according to the invention can also be eucaryoteor procaryote cells. Among the eucaryote cells which are suitable, canbe cited animal cells, yeasts, or mushrooms. In particular, for yeasts,can be cited the yeasts of the type Saccharomyces, Kluyveromyces,Pichia, Schwanniomyces, or Hansenula. As to animal cells, can be citedthe cells COS, CHO, C127, NIH-3T3, etc. For mushrooms can be cited moreparticularly Aspergillus ssp. or Trichoderma ssp. As procaryote hosts,it is preferred to use the following bacteria: E.coli, Bacillus, orStreptomyces. The cells thus obtained can be used for measuring thecapacity of different molecules to be used as ligands or as modulatorsof the activity of the opioid receptors. More particularly, they canthus be used in a procedure for elaborating and isolating ligands or formodulating the activity of opioid receptors, and more preferably,agonists and antagonists.

Another object of the invention hence relates to the process ofelaborating and/or isolating ligands of the opioid receptors, accordingto which the following steps are performed:

there is placed in contact a molecule or a mixture containing differentmolecules, if desired unidentified, with a recombinant cell such asdescribed above expressing at its surface a polypeptide having an opioidreceptor activity under conditions permitting interaction between saidpolypeptide and said molecule in the case in which the latter has anaffinity for said polypeptide, and

detecting and/or isolating the molecules bound to said polypeptide.

In a particular embodiment, this process of the invention is adapted forthe elaboration and/or isolation of agonists and antagonists ofdynorphine or other ligands of the kappa receptors for the kappa opioidreceptors.

Another object of the invention relates to a process for elaboratingand/or isolating modulators of the opioid receptors, according to whichthe following steps are performed:

a molecule or a mixture containing different molecules, if desiredunidentified, is placed in contact with a recombinant cell such asdescribed above expressing at its surface a polypeptide having an opioidreceptor activity, in the presence of the endogenous ligand of saidreceptor, under conditions permitting interaction between saidpolypeptide and its ligand, and

there are detected and/or isolated the molecules capable of modulatingthe activity of the ligand on said polypeptide.

In a particular embodiment, this process of the invention is adapted forthe elaboration and/or isolation of modulators of the activity ofdynorphine or other ligands of the kappa receptors on the kappa opioidreceptors.

Another object of the invention is the use of a ligand or a modulatoridentified and/or obtained according to the process described above, asa medication. Such ligands or modulators can thus permit treatingcertain afflictions connected to the opioid receptors. In particular,the kappa opioid receptors being mediators of an analgesic effect, theagonists of these receptors can be used to decrease the sensations ofpain.

The invention also relates to any medication comprising as activeprinciple at least one molecule acting on a receptor of the invention.Preferably, the molecule is a ligand or a modulator identified and/orisolated according to the process described above.

Other advantages of the present invention will become apparent from areading of the examples which follow, which are to be considered asillustrative and not limiting.

DESCRIPTION OF THE FIGURES

FIG. 1: Partial organization of the gene of the human kappa opioidreceptor (hKOR). The exons are indicated by boxes and the introns bylines. The position of the putative transmembranal domains of the kappareceptor are indicated by Roman numerals. The known ends of the exonsare numerically designated by Arabic numerals, relative to theirposition in cDNA (FIG. 2). The exon in 3' has been sequenced to theterminal codon TGA.

FIG. 2: Strategy for cloning cDNA coding for the human kappa opioidreceptor.

GENERAL TECHNIQUES FOR CLONING

Conventional methods used in molecular biology such as preparativeextractions of plasmid DNA, the centrifugation of plasmid DNA in agradient of cesium chloride, electrophoresis on agarose or acrylamidegels, purification of DNA fragments by electroelution, extractions ofproteins with phenol or phenol-chloroform, the precipitation of DNA insaline medium by ethanol or isopropanol, transformation with Escherichiacoli, etc., are well known to those skilled in the art and areplentifully described in the literature [Maniatis T. et al., "MolecularCloning, a Laboratory Manual", Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. 1982; Ausubel F. M. et al. (eds), "Current Protocolsin Molecular Biology", John Wiley & Sons, New York, 1987].

For binding, the DNA fragments are separated according to their size byelectrophoresis on agarose or acrylamide gels, extracted with phenol orby a phenol/chloroform mixture, precipitated in ethanol, then incubatedin the presence of DNA ligase of phage T4 according to the directions ofthe supplier.

Filling the protruding ends 5' is effected by the Klenow fragment of DNAof Polymerase. I E.coli according to the directions of the supplier. Thedestruction of the projecting ends 3' is effected in the presence of DNAPolymerase of T4 phage utilized according to the instructions of thesupplier. The destruction of the projecting 5' ends is carried out by atreatment provided by S1 nuclease.

Mutagenesis directed in vitro by synthetic oligode-oxynucleotides iscarried out according to the method developed by Taylor et al. [NucleicAcids Res. 13 (1985) 8749-8764].

Enzymatic amplification of the DNA fragments is carried by the so-calledPCR technique [Polymerase-catalyzed Chain Reaction, Saiki R. K. et al.,Science 230 (1985) 1350-1354; Mullis K. B. and Faloona F. A., Meth.Enzym. 155 (1987) 335-350].

Verification of the nucleotide sequences is carried out by the methoddeveloped by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977)5463-5467].

For hybridation experiments, the control conditions are based onManiatis T. et al., above.

1. Genomic Cloning and Partial Organization of the Gene

A human genome bank has been scanned with the help of a cDNA probecoding for the delta receptor of mice (Kieffer et al., PNAS 1992, Vol.89, 12048). The probe is a cDNA fragment Pst I-Not I (976 bp) whichcorresponds to most of the coding portion of cDNA. The bank was spreadout and transferred onto nitrocellulose filters. These latter werehybridized with a probe marked with ³² P, under relatively stringentconditions (5×SSC, 5×Denhardt's, SDS 0.1%, Formamide 40%, NaPPi 0.05%and DNA of salmon sperm 100 μg/ml). Washing was carried out with 0.1%SSC at 50° C. and the filters exposed to an X-ray film overnight.

Two genomic clones hybridizing weakly with the probe were isolated. Theanalysis of the inserts by partial sequencing indicates that theycontain regions very homologous to the kappa receptor of mice. An exonwas identified in each clone: one codes for the correspondingtopological region at the beginning of the first intracellular loop tothe end of the second transmembranal domain, and the other codes for therest of the C-terminal end of the receptor. The topology of this portionof the gene is shown in FIG. 1 and the exact position of the intron-exonjunctions is indicated thereon.

2. Cloning of a Complete cDNA Coding for the Human Kappa Receptor

Most of the methods used ("standard"), except recited in the text,derive from Sambrook, J. et al., 1989 Molecular Cloning: A LaboratoryManual (Cold Spring Harbor Lab., Cold Spring Harbor, N.Y.), 2nd Ed.

The human placenta expresses the kappa receptor (Meunier et al., 1988Life Sci., Vol. 43, 559). We have prepared the total RNA of the humanplacenta (Chomczynski, P. et al., 1987, Anal. Biochem., Vol. 162, 156)and have synthesized cDNA from this RNA, with the help of a randomnucleotide trigger ("random hexamer", Pharmacia) and with the aid ofMoloney Murine Leukemia Virus reverse transcriptase (BRL), understandard conditions.

We have then amplified the cDNA coding for the human kappa receptor byPCR (Polymerase Chain Reaction) by using portions of the human sequenceobtained from the analysis of the genomic clones for the conception ofspecific triggers. THE 5' end of the coding portion of the gene beinglacking, we have used an oligonucleotide derived from a kappa sequenceof mice, for amplification from the end 5' end.

The use of two triggers located at the 5' (mice) and 3' (human) terminiof the coding region being apparently less effective for the productionof a complete cDNA, we have proceeded in three steps and the strategyused is shown in FIG. 2 (the figures indicate the precise position ofthe nucleotides in the complete sequence of cDNA shown in the sequenceSEQ ID No. 1).

The sequence of the oligonucleotides utilized is as follows:

RP69: 5' GAGAGCTCGCGGCCGCGAGCTGCAGCGCTCACCATG (SEQ ID No. 2)

RH84: 5' CACGGGGTGGCACACGGCAA (SEQ ID No. 4)

RN6: 5' GTCTACTFGATGAATWCCTGG (SEQ ED No. 5)

RP70: 5' AGACCCAAGCTTGCCCGGGTCCACGACTAGTCATACTGG (SEQ ID No. 6)

The sequences hybridizing to cDNA are indicated in fat, the othernucleotides being present to facilitate the ultimate step of cloning thefragments produced by the reaction of PCR.

The oligonucleotides RP70, RH84 and RN6 are derived from the human kappasequence. The oligonucleotide RP69 corresponds to a sequence 5' notcoding for mice (including the ATG initiator at the 3' end) so as not tointroduce a murine sequence in human cDNA. The precise position of theregions of cDNA which hybridize to the four triggers is indicated inFIG. 2

The different amplification reactions have been carried out under thefollowing conditions:

Reaction 1: Triggers RN6 and RP70. The reaction is carried out in thepresence of Taq Polymerase (Cetus) under standard PCR conditions, exceptfor the addition of 5% DMSO into the incubation medium. Theamplification takes place for 40 cycles (1 minute at 94° C., 1 minute at55° C., 1 minute at 72° C.). The last stage of elongation is carried outfor 10 minutes at 720C. There is thus obtained A DNA fragment of thedesired size (760 bp)=fragment 1.

Reaction 2: The amplification conditions are the same as for reaction 1.Two successive amplifications were necessary: the first uses thetriggers RP69 and RP70 and leads to the obtention of a mixture offragments of DNA of a size comprised between 1 and 1.3 kb, containingcomplete cDNA. This mixture is purified after electrophoresis on a 1%agarose gel by the GeneClean process and reamplified with the aid ofoligonucleotides RP69 and RH84. There is thus obtained a fragment of DNAof a desired size (508 bp)=fragment 2.

Cloning of Complete cDNA:

The PCR fragments are stitched and cloned in the free ends of thepBluescript vector (Strategene) linearized with EcoR V. The inserts aresequenced in the two directions on an automatic DNA sequencer (373A DNA,Applied Biosystems Inc.). Then the two inserts are excised from theplasmid in the presence of EcoR I (cut side 3' of fragment 2 and side 5'of fragment 1) and of a restriction enzyme of the polylinker. Thefragments are purified with 1% agarose gel by the GeneClean process andcoligated in the vector pcDNA/Amp (Invitrogen) for the transitoryexpression of the receptor of Cos cells.

The two amplification reactions have produced the desired fragments 1and 2. In the case of fragment 2, the use of an oligonucleotide derivedfrom a mouse series at end 5' has permitted the amplification of humancDNA.

The two fragments cover themselves on 140 bp. The covering sequence isidentical for the two fragments. The sequences of the two fragments arecompared to the predetermined sequence from genomic DNA (position 257 toposition 1143) and found to be perfectly identical. Concerning theportion of sequence 1 to 256, whose genome sequence is unknown to us, wehave verified that the amplification with PCR has not introduced errors:we have compared the sequence of fragment 2 obtained from the threedifferent amplification reactions and have found them identical in thethree cases.

The cDNA sequence coding for the human kappa receptor according to theinvention, as well as the derived protein sequence (SEQ ID NO: 2), arepresent in the sequence SEQ ID No. 1.

3. Conclusion: Primary Structure of the Human Kappa Receptor

We have used a probe coding for a delta opioid rector in mice so as toscan a human genome bank carefully and to isolate the homologous DNAgenome fragments.

We have identified two exons which by comparison of sequence, codeapparently for the human kappa opioid receptor. We have isolated an cDNAdrive from this gene (coding portion) from a human tissue (placenta) andwe have analyzed its primary structure (SEQ ID No. 1).

cDNA has a size of 1101 base pairs and codes for a protein with 380amino acids. The nucleotide sequence is homologous to 86.7% to mousecDNA coding for a kappa opioid receptor. The greatest regions ofdivergence are situated in the regions of the N- and C-terminals. Thesesame regions are also the least conserved regions between the threesubtypes mu, delta and kappa.

4. Pharmacological Study of the Receptor K56

The vector pcDNA/Amp containing the cDNA coding for the opioid receptoraccording to the invention isolated in Example 2 is used to transfectCos-1 cells. The membranes of the transfected cells obtained are thenprepared and tested for their capacity to bind certain marked opioidligands.

The plasmid vector pcDNA/Amp purified with cesium chloride is used totransfect the Cos-1 cells by using the technique of DEAE-dextran.

72 hours after transfection, the recombinant cells are gathered and themembranes are prepared in the following manner: the cellular bottoms aregathered, at 4° C., in 60 ml of a buffer Tris-HCl 50 mM pH 7.4; EDTA 10mM, homogenized and centrifuged at 1100 g for 10 minutes. The base isthen taken up a second time in 30 ml of the same buffer, homogenized andcentrifuged. The 2 supernatants are combined and centrifuged at 110,000g for 15 minutes. The membranous base is then taken up in 5 ml of thesame buffer, aliquoted and stored at -80° C. The experiments for acombination at saturation and for competition are then carried out onthese membranes in the presence of different ligands. For that, thespecimens of membrane (15-30 μg of proteins) are incubated 2 hours at25° C. in the presence of ³ H-diprenorphine or ³ H-U-69,593, with orwithout a competitor, in a final volume of 1 ml of buffer Tris-HCl 50 mM(pH 7.4); EDTA 10 mM. The reaction is then stopped by filtration undervacuum on Whatman GF/B filters, and rinsed 3 times with 3 ml of coldbuffer. The values of Ki are obtained by following the equation of Chengand Prusso Ki=IC50/(1+L/Kd). The radioactivity has been measured with aβ counter.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 6                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1142 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1142                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - ATG GAC TCC CCG ATC CAG ATC TTC CGC GGG GA - #G CCG GGC CCT ACC        TGC       48                                                                    Met Asp Ser Pro Ile Gln Ile Phe Arg Gly Gl - #u Pro Gly Pro Thr Cys            1               5 - #                 10 - #                 15              - - GCC CCG AGC GCC TGC CTG CCC CCC AAC AGC AG - #C GCC TGG TTT CCC GGC           96                                                                       Ala Pro Ser Ala Cys Leu Pro Pro Asn Ser Se - #r Ala Trp Phe Pro Gly                        20     - #             25     - #             30                  - - TGG GCC GAG CCC GAC AGC AAC GGC AGC GCC GG - #C TCG GAG GAC GCG CAG          144                                                                       Trp Ala Glu Pro Asp Ser Asn Gly Ser Ala Gl - #y Ser Glu Asp Ala Gln                    35         - #         40         - #         45                      - - CTG GAG CCC GCG CAC ATC TCC CCG GCC ATC CC - #G GTC ATC ATC ACG GCG          192                                                                       Leu Glu Pro Ala His Ile Ser Pro Ala Ile Pr - #o Val Ile Ile Thr Ala                50             - #     55             - #     60                          - - GTC TAC TCC GTA GTG TTC GTC GTG GGC TTG GT - #G GGC AAC TCG CTG GTC          240                                                                       Val Tyr Ser Val Val Phe Val Val Gly Leu Va - #l Gly Asn Ser Leu Val            65                 - # 70                 - # 75                 - # 80       - - ATG TTC GTG ATC ATC CGA TAC ACA AAG ATG AA - #G ACA GCA ACC AAC ATT          288                                                                       Met Phe Val Ile Ile Arg Tyr Thr Lys Met Ly - #s Thr Ala Thr Asn Ile                            85 - #                 90 - #                 95              - - TAC ATA TTT AAC CTG GCT TTG GCA GAT GCT TT - #A GTT ACT ACA ACC ATG          336                                                                       Tyr Ile Phe Asn Leu Ala Leu Ala Asp Ala Le - #u Val Thr Thr Thr Met                       100      - #           105      - #           110                  - - CCC TTT CAG AGT ACG GTC TAC TTG ATG AAT TC - #C TGG CCT TTT GGG GAT          384                                                                       Pro Phe Gln Ser Thr Val Tyr Leu Met Asn Se - #r Trp Pro Phe Gly Asp                   115          - #       120          - #       125                      - - GTG CTG TGC AAG ATA GTA ATT TCC ATT GAT TA - #C TAC AAC ATG TTC ACC          432                                                                       Val Leu Cys Lys Ile Val Ile Ser Ile Asp Ty - #r Tyr Asn Met Phe Thr               130              - #   135              - #   140                          - - AGC ATC TTC ACC TTG ACC ATG ATG AGC GTG GA - #C CGC TAC ATT GCC GTG          480                                                                       Ser Ile Phe Thr Leu Thr Met Met Ser Val As - #p Arg Tyr Ile Ala Val           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - TGC CAC CCC GTG AAG GCT TTG GAC TTC CGC AC - #A CCC TTG AAG GCA        AAG      528                                                                    Cys His Pro Val Lys Ala Leu Asp Phe Arg Th - #r Pro Leu Lys Ala Lys                          165  - #               170  - #               175              - - ATC ATC AAT ATC TGC ATC TGG CTG CTG TCG TC - #A TCT GTT GGC ATC TCT          576                                                                       Ile Ile Asn Ile Cys Ile Trp Leu Leu Ser Se - #r Ser Val Gly Ile Ser                       180      - #           185      - #           190                  - - GCA ATA GTC CTT GGA GGC ACC AAA GTC AGG GA - #A GAC GTC GAT GTC ATT          624                                                                       Ala Ile Val Leu Gly Gly Thr Lys Val Arg Gl - #u Asp Val Asp Val Ile                   195          - #       200          - #       205                      - - GAG TGC TCC TTG CAG TTC CCA GAT GAT GAC TA - #C TCC TGG TGG GAC CTC          672                                                                       Glu Cys Ser Leu Gln Phe Pro Asp Asp Asp Ty - #r Ser Trp Trp Asp Leu               210              - #   215              - #   220                          - - TTC ATG AAG ATC TGC GTC TTC ATC TTT GCC TT - #C GTG ATC CCT GTC CTC          720                                                                       Phe Met Lys Ile Cys Val Phe Ile Phe Ala Ph - #e Val Ile Pro Val Leu           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - ATC ATC ATC GTC TGC TAC ACC CTG ATG ATC CT - #G CGT CTC AAG AGC        GTC      768                                                                    Ile Ile Ile Val Cys Tyr Thr Leu Met Ile Le - #u Arg Leu Lys Ser Val                          245  - #               250  - #               255              - - CGG CTC CTT TCT GGC TCC CGA GAG AAA GAT CG - #C AAC CTG CGT AGG ATC          816                                                                       Arg Leu Leu Ser Gly Ser Arg Glu Lys Asp Ar - #g Asn Leu Arg Arg Ile                       260      - #           265      - #           270                  - - ACC AGA CTG GTC CTG GTG GTG GTG GCA GTC TT - #C GTC GTC TGC TGG ACT          864                                                                       Thr Arg Leu Val Leu Val Val Val Ala Val Ph - #e Val Val Cys Trp Thr                   275          - #       280          - #       285                      - - CCC ATT CAC ATA TTC ATC CTG GTG GAG GCT CT - #G GGG AGC ACC TCC CAC          912                                                                       Pro Ile His Ile Phe Ile Leu Val Glu Ala Le - #u Gly Ser Thr Ser His               290              - #   295              - #   300                          - - AGC ACA GCT GCT CTC TCC AGC TAT TAC TTC TG - #C ATC GCC TTA GGC TAT          960                                                                       Ser Thr Ala Ala Leu Ser Ser Tyr Tyr Phe Cy - #s Ile Ala Leu Gly Tyr           305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - ACC AAC AGT AGC CTG AAT CCC ATT CTC TAC GC - #C TTT CTT GAT GAA        AAC     1008                                                                    Thr Asn Ser Ser Leu Asn Pro Ile Leu Tyr Al - #a Phe Leu Asp Glu Asn                          325  - #               330  - #               335              - - TTC AAG CGG TGT TTC CGG GAC TTC TGC TTT CC - #A CTG AAG ATG AGG ATG         1056                                                                       Phe Lys Arg Cys Phe Arg Asp Phe Cys Phe Pr - #o Leu Lys Met Arg Met                       340      - #           345      - #           350                  - - GAG CGG CAG AGC ACT AGC AGA GTC CGA AAT AC - #A GTT CAG GAT CCT GCT         1104                                                                       Glu Arg Gln Ser Thr Ser Arg Val Arg Asn Th - #r Val Gln Asp Pro Ala                   355          - #       360          - #       365                      - - TAC CTG AGG GAC ATC GAT GGG ATG AAT AAA CC - #A GTA  TG                  - #   1142                                                                    Tyr Leu Arg Asp Ile Asp Gly Met Asn Lys Pr - #o Val                               370              - #   375              - #   380                          - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 380 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Asp Ser Pro Ile Gln Ile Phe Arg Gly Gl - #u Pro Gly Pro Thr Cys        1               5 - #                 10 - #                 15              - - Ala Pro Ser Ala Cys Leu Pro Pro Asn Ser Se - #r Ala Trp Phe Pro Gly                   20     - #             25     - #             30                  - - Trp Ala Glu Pro Asp Ser Asn Gly Ser Ala Gl - #y Ser Glu Asp Ala Gln               35         - #         40         - #         45                      - - Leu Glu Pro Ala His Ile Ser Pro Ala Ile Pr - #o Val Ile Ile Thr Ala           50             - #     55             - #     60                          - - Val Tyr Ser Val Val Phe Val Val Gly Leu Va - #l Gly Asn Ser Leu Val       65                 - # 70                 - # 75                 - # 80       - - Met Phe Val Ile Ile Arg Tyr Thr Lys Met Ly - #s Thr Ala Thr Asn Ile                       85 - #                 90 - #                 95              - - Tyr Ile Phe Asn Leu Ala Leu Ala Asp Ala Le - #u Val Thr Thr Thr Met                  100      - #           105      - #           110                  - - Pro Phe Gln Ser Thr Val Tyr Leu Met Asn Se - #r Trp Pro Phe Gly Asp              115          - #       120          - #       125                      - - Val Leu Cys Lys Ile Val Ile Ser Ile Asp Ty - #r Tyr Asn Met Phe Thr          130              - #   135              - #   140                          - - Ser Ile Phe Thr Leu Thr Met Met Ser Val As - #p Arg Tyr Ile Ala Val      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Cys His Pro Val Lys Ala Leu Asp Phe Arg Th - #r Pro Leu Lys Ala        Lys                                                                                             165  - #               170  - #               175             - - Ile Ile Asn Ile Cys Ile Trp Leu Leu Ser Se - #r Ser Val Gly Ile Ser                  180      - #           185      - #           190                  - - Ala Ile Val Leu Gly Gly Thr Lys Val Arg Gl - #u Asp Val Asp Val Ile              195          - #       200          - #       205                      - - Glu Cys Ser Leu Gln Phe Pro Asp Asp Asp Ty - #r Ser Trp Trp Asp Leu          210              - #   215              - #   220                          - - Phe Met Lys Ile Cys Val Phe Ile Phe Ala Ph - #e Val Ile Pro Val Leu      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ile Ile Ile Val Cys Tyr Thr Leu Met Ile Le - #u Arg Leu Lys Ser        Val                                                                                             245  - #               250  - #               255             - - Arg Leu Leu Ser Gly Ser Arg Glu Lys Asp Ar - #g Asn Leu Arg Arg Ile                  260      - #           265      - #           270                  - - Thr Arg Leu Val Leu Val Val Val Ala Val Ph - #e Val Val Cys Trp Thr              275          - #       280          - #       285                      - - Pro Ile His Ile Phe Ile Leu Val Glu Ala Le - #u Gly Ser Thr Ser His          290              - #   295              - #   300                          - - Ser Thr Ala Ala Leu Ser Ser Tyr Tyr Phe Cy - #s Ile Ala Leu Gly Tyr      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Thr Asn Ser Ser Leu Asn Pro Ile Leu Tyr Al - #a Phe Leu Asp Glu        Asn                                                                                             325  - #               330  - #               335             - - Phe Lys Arg Cys Phe Arg Asp Phe Cys Phe Pr - #o Leu Lys Met Arg Met                  340      - #           345      - #           350                  - - Glu Arg Gln Ser Thr Ser Arg Val Arg Asn Th - #r Val Gln Asp Pro Ala              355          - #       360          - #       365                      - - Tyr Leu Arg Asp Ile Asp Gly Met Asn Lys Pr - #o Val                          370              - #   375              - #   380                          - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - GAGAGCTCGC GGCCGCGAGC TGCAGCGCTC ACCATG      - #                  -     #       36                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - CACGGGGTGG CACACGGCAA            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - GTCTACTTGA TGAATTCCTG G           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - AGACCCAAGC TTGCCCGGGT CCACGACTAG TCATACTGG      - #                      - #    39                                                                    __________________________________________________________________________

What is claimed is:
 1. An isolated and purified nucleic acid comprisinga nucleotide sequence selected from the group consisting of:SEQ ID NO:1,and its complementary strand.
 2. The nucleic acid according to claim 1,further comprising a sequence encoding a signal peptide.
 3. A host celltransfected or transformed with the nucleic acid of claim 1 comprisingSEO ID NO:2.
 4. A method for detecting ligands that bind to kappa opioidreceptors, comprising the following steps:contacting a test opioidligand with a host cell according to claim 3, wherein said cellexpresses a human kappa opioid receptor at its surface, under conditionswhich permit interaction between said polypeptide and said test opioidligand, and detecting an opioid ligand bound to said polypeptide.
 5. Anisolated kappa opioid receptor free from other naturally-occurringproteins and comprising SEQ ID NO:2.